β-Galactosidase (β-gal), an important enzyme used in the food industry to hydrolyze milk and whey, was immobilized onto chitosan microparticles prepared by the emulsion crosslinking method. The immobilization conditions such as enzyme binding capacity of the microparticles, crosslinking agent concentration, and immobilization time were optimized. A 3% glutaraldehyde concentration was found as optimal and allowed an enzyme loading of 27 mg protein/g dry support. Reduction with sodium borohydride after the covalent attachment was beneficial for the catalytic activity, leading to a total activity yield of 23.5% related to the native Kluyveromyces lactis β-gal subjected to immobilization. The immobilized biocatalyst was characterized by pH dependence, thermostability and distribution of protein by FITC labeling.